The long term objective of this proposal is the identification of genes involved in the regulation of limb development through the study of developmental defects affecting the limbs. Two such defects are the Split Hand-Split Foot malformation and Acheiropodia which manifest median clefts and absence of hand feet and forearms respectively. In our previous studies we mapped a Split Hand-Split Foot locus on 10q24 (SHFM3) and predicted the existence of at least one additional locus. We have narrowed the SHFM3 critical distance and identified a candidate gene. During the time period of the proposed project we intend to focus on the following objectives: 1) To define the molecular basis of SHFM3 by obtaining the entire coding sequence of the candidate gene, obtain its expression pattern in developing mouse embryos, screen for mutations in individuals affected with SHFM3. 2) To map the new autosomal locus for Split Hand Split Food (SHFM4) by whole genome search. We will genotype our family panel with highly polymorphic microsatellite repeat markers evenly distributed in the genome. Initial evidence of linkage will be followed by saturation mapping and multipoint linkage analysis. 3) To map the acheiropodia locus by whole genome search using homozygosity mapping. By elucidating the molecular basis of SHFM3 and mapping SHFM4 and Acheiropodia we will have identified at least three genes playing a critical role in limb development.